Alterations in gene transcription may cause changes in plasma lipid and lipoprotein levels and in some instances increase the risk of cardiovascular disease. The long-term objective of the proposed studies is to understand the mechanisms of transcriptional regulation of the human apolipoprotein genes. This line of research which we initiate in 1990 was based on the hypothesis that the regulation of transcription of apolipoprotein genes is controlled by protein-protein interaction of tissue-specific and ubiquitous factors which recognize the apolipoprotein promoters as well as interaction of these factors with bridging proteins and other components of the basal transcription system. During the last 3 years we have made strides in understanding the complex organization of 5 apolipoprotein promoters (apoA-I, apoCIII, apoA-IV, apoA-II & apoB) and several of the factors which are involved in the regulation of the corresponding genes. Based on this broad perspective we have acquired from our own work and the progress in the field, our current specific aims are: 1) To characterize fully the SREBP related clones we have identified by screening cDNA libraries and study their functions and their role in the regulation of the human apoCIII and apoA-II genes. 2) To study the mechanism of transcriptional regulation of the apoA-I, apoCIII gene complex in cell cultures. This will include a) study of the role of nuclear receptors for retinoids, thyroids and peroxisome proliferators which bind to the HRBs of the apoA-I and apoCIII promoters and their ligands on the transcriptional regulation of the apoA-I, apoCIII genes. b) Study of the role of SP1 or related proteins which bind on the apoCIII enhancer on the transcriptional regulation of the apoA-I and apoCIII genes. These studies will utilize in vitro DNA binding and supershift assays, cell transfection and in vitro transcription assays. The in vivo physiological importance of SP1 and RXRalpha will be studied by inhibition of their synthesis using antisense methodology. 3) To define by in vivo footprinting (LM-PCR) the regulatory regions of the proximal apoA-I and apoCIII promoter as well as the apoCIII enhancer which are important for tissue specific transcription in vivo. 4) To study by transgenic methodologies a) the role of the apoCIII enhancer on the tissue specific transcription of the human apoCIII and apoA-I genes and its ability to promote intestinal transcription in vivo. b) The role of the HRE and SP1 sites on the transcriptional regulation of the human apoCIII and apoA-I genes in vivo. It is expected that the proposed studies will provide new insights into the mechanism of transcriptional regulation of the apoA-I and apoCIII genes as well as general insights into hepatic and intestinal gene regulation. Increases in plasma apoA-I and HDL concentration are associated with protection from cardiovascular disease. Alteration in apoCIII is thought to affect the catabolism of triglyceride-rich lipoproteins. Thus the information obtained from this project may provide rational approaches towards correcting low plasma HDL levels and hypertriglyceridemia.